Pluripotent embryonic stem cells represent a promising renewable cell source to generate a variety of differentiated cell types. Although many investigators have described techniques to effectively differentiate stem cells into different mature cell lineages, their practicality is limited by the absence of large scale processing consideration and low yields of differentiated cells. Previously we have established a murine embryonic stem cell alginate-poly-l-lysine microencapsulation differentiation system. The three-dimensional alginate microenvironment maintains cell viability, is conducive to ES cell differentiation to hepatocyte lineage cells, and maintains differentiated cellular function. In the present work, we demonstrate that hepatocyte differentiation is mediated by cell-cell aggregation in the encapsulation microenvironment. Both cell aggregation and hepatocyte functions, such as urea and albumin secretion, as well as increased expression of cytokaratin 18 and cyp4507a, occur concomitantly with surface E-cadherin expression. Furthermore, by incorporating soluble inducers, such as retinoic acid, into the permeable microcapsule system, we demonstrate decreased cell aggregation and enhanced neuronal lineage differentiation with the expression of various neuronal specific markers, including neurofilament, A2B5, O1 and GFAP. Therefore, as a result of capsule parameter and microenvironment manipulation, we are capable of targeting cellular differentiation to both endodermal and ectodermal cell lineages.

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